Methods for preserving organs and tissues

ABSTRACT

The invention relates to a method for preserving an organ or tissue comprising contacting the organ or tissue with an effective amount of a kallikrein inhibitor and solutions useful for such a method. Also provided is a method for reducing reperfusion injury of an organ during surgery and/or following removal of the organ from a subject comprising placing the organ in an organ storage and preservative solution, wherein the solution comprises a kallikrein inhibitor.

RELATED APPLICATION

This application is a continuation (and claims the benefit of priorityunder 35 USC 120) of U.S. application Ser. No. 11/621,246, filed Jan. 9,2007, which is a divisional (and claims the benefit of priority under 35USC 120) of U.S. application Ser. No. 10/456,981, filed Jun. 6, 2003,now U.S. Pat. No. 7,166,576, which claims the benefit of U.S.Provisional application Ser. No. 60/407,004, filed Aug. 28, 2002. Thedisclosure of the prior applications are considered part of (and isincorporated by reference in) the disclosure of this application.

BACKGROUND OF THE INVENTION

Preservation of the viability of donor organs is an important goal fororgan transplantation. Typically the organ to be transplanted must bestored and shipped to the prospective recipient. The ability to prolongthe cellular viability of the organ during storage and transportation isvery important to the success of the transplant operation. Preservativesolutions play an important role in the longevity of the organ.Solutions for organ preservation include those described by Berdyaev etal., U.S. Pat. No. 5,432,053; Belzer et al., U.S. Pat. Nos. 4,798,824,4,879,283, and 4,873,230; Taylor, U.S. Pat. No. 5,405,742; Dohi et al.,U.S. Pat. No. 5,565,317; Stern et al., U.S. Pat. Nos. 5,370,989 and5,552,267, the contents of which are incorporated herein by reference intheir entirety. However, a need exists for improved methods andsolutions for organ preservation. Proteases are involved in a broadrange of biological pathways. In particular, serine proteases such askallikrein, plasmin, elastase, urokinase plasminogen activator,thrombin, human lipoprotein-associated coagulation inhibitor, andcoagulation factors such as factors VIIa, IXa, Xa, XIa, and XIIa havebeen implicated in pathways affecting blood flow, e.g., general andfocal ischemia, tumor invasion, fibrinolysis, perioperative blood loss,and inflammation. Inhibitors of specific serine proteases, therefore,have received attention as potential drug targets for various ischemicmaladies.

One such inhibitor, aprotinin (also called bovine pancreatic trypsininhibitor or BPTI), obtained from bovine lung, has been approved in theUnited States for prophylactic use in reducing perioperative blood lossand the need for transfusion in patients undergoing CPB, e.g., in thecourse of a coronary artery bypass grafting procedure. Aprotinin iscommercially available under the trade name TRASYLOL® (Bayer CorporationPharmaceutical Division, West Haven, Conn.) and was previously approvedfor use to treat pancreatitis. The effectiveness of aprotinin isassociated with its relatively non-specific abilities to inhibit avariety of serine proteases, including plasma kallikrein, and plasmin.These proteases are important in a number of pathways of the contactactivation system (CAS).

CAS is initially activated when whole blood contacts the surface offoreign substrates (e.g., kaolin, glass, dextran sulfate, or damagedbone surfaces). Kallikrein, a serine protease, is a plasma enzyme thatinitiates the CAS cascade leading to activation of neutrophils, plasmin,coagulation, and various kinins. Kallikrein is secreted as a zymogen(pre-kallikrein) that circulates as an inactive molecule until activatedby a proteolytic event early in the contact activation cascade.

However, the use of specific kallikrein inhibitors for organpreservation has not been successfully demonstrated.

SUMMARY OF THE INVENTION

This invention is based on the discovery of peptides that inhibit serineproteases, such as, for example, kallikrein, which can successfully beemployed to preserve an organ pending transplant. More specifically, theinvention provides methods of using kallikrein inhibitors in a methodfor preserving an organ or tissue and compositions for such use. Theinvention also relates to methods for reducing, inhibiting or preventingreperfusion injury or damage in an organ or tissue that has been removedfrom its host and compositions for such use. Preferred kallikreinpeptides include those described in U.S. Pat. Nos. 6,333,402 and6,057,287 to Markland et al., the contents of which are incorporatedherein by reference in their entirety.

In a particularly preferred embodiment, the invention is directed tocompositions comprising a polypeptide comprising the amino acidsequence:

(SEQ ID NO: 1) Xaa1 Xaa2 Xaa3 Xaa4 Cys Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11Gly Xaa13 Cys Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 Xaa23Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Cys Xaa31 Xaa32 Phe Xaa34 Xaa35 GlyGly Cys Xaa39 Xaa40 Xaa41 Xaa42 Xaa43 Xaa44 Xaa45 Xaa46 Xaa47 Xaa48Xaa49 Xaa50 Cys Xaa52 Xaa53 Xaa54 Cys Xaa56 Xaa57 Xaa58,wherein Xaa1, Xaa2, Xaa3, Xaa4, Xaa56, Xaa57 or Xaa58 are eachindividually an amino acid or absent; Xaa6, Xaa7, Xaa8, Xaa9, Xaa20,Xaa24, Xaa25, Xaa26, Xaa27, Xaa28, Xaa29, Xaa41, Xaa42, Xaa44, Xaa46,Xaa47, Xaa48, Xaa49, Xaa50, Xaa52, Xaa53 and Xaa54 can be any aminoacid; Xaa10 is an amino acid selected from the group consisting of: Aspand Glu; Xaa11 is an amino acid selected from the group consisting of:Asp, Gly, Ser, Val, Asn, Ile, Ala and Thr; Xaa13 is an amino acidselected from the group consisting of: Arg, His, Pro, Asn, Ser, Thr,Ala, Gly, Lys and Gln; Xaa15 is an amino acid selected from the groupconsisting of: Arg, Lys, Ala, Ser, Gly, Met, Asn and Gln; Xaa16 is anamino acid selected from the group consisting of: Ala, Gly, Ser, Asp andAsn; Xaa17 is an amino acid selected from the group consisting of: Ala,Asn, Ser, Ile, Gly, Val, Gln and Thr; Xaa18 is an amino acid selectedfrom the group consisting of: His, Leu, Gln and Ala; Xaa19 is an aminoacid selected from the group consisting of: Pro, Gln, Leu, Asn and Ile;Xaa21 is an amino acid selected from the group consisting of: Trp, Phe,Tyr, His and Ile; Xaa22 is an amino acid selected from the groupconsisting of: Tyr and Phe; Xaa23 is an amino acid selected from thegroup consisting of: Tyr and Phe; Xaa31 is an amino acid selected fromthe group consisting of: Glu, Asp, Gln, Asn, Ser, Ala, Val, Leu, Ile andThr; Xaa32 is an amino acid selected from the group consisting of: Glu,Gln, Asp Asn, Pro, Thr, Leu, Ser, Ala, Gly and Val; Xaa34 is an aminoacid selected from the group consisting of: Thr, Ile, Ser, Val, Ala,Asn, Gly and Leu; Xaa35 is an amino acid selected from the groupconsisting of: Tyr, Trp and Phe; Xaa39 is an amino acid selected fromthe group consisting of: Glu, Gly, Ala, Ser and Asp; Xaa40 is an aminoacid selected from the group consisting of: Gly and Ala; Xaa43 is anamino acid selected from the group consisting of: Asn and Gly; Xaa45 isan amino acid selected from the group consisting of: Phe and Tyr; andwherein said polypeptide inhibits kallikrein, and methods of using suchcompositions.

In a particular embodiment, specific amino acid positions can be thefollowing: Xaa6 can be Ala, Xaa7 can be Phe, Xaa8 can be Lys, Xaa9 canbe Ala, Xaa10 can be Asp, Xaa11 can be Asp, Xaa13 can be Pro, Xaa15 canbe Arg, Xaa16 can be Ala, Xaa17 can be Ala, Xaa18 can be His, Xaa19 canbe Pro, Xaa20 can be Arg, Xaa24 can be Asn, Xaa25 can be Ile, Xaa26 canbe Phe, Xaa27 can be Thr, Xaa28 can be Arg, Xaa29 can be Gln, Xaa31 canbe Glu, Xaa32 can be Glu, Xaa34 can be Ile, Xaa35 can be Tyr, Xaa39 canbe Glu, Xaa41 can be Asn, Xaa42 can be Arg, Xaa44 can be Arg, Xaa46 canbe Glu, Xaa47 can be Ser, Xaa48 can be Leu, Xaa49 can be Glu, and/orXaa50 can be Glu; any of these specific amino acids at these positionscan occur individually or in combination with one or more of the aminoacids at one or more position otherwise described.

In a particular embodiment, the present invention is directed to acomposition comprising a polypeptide as described in SEQ ID NO:1, suchthat two or more of the following amino acid positions are defined asfollows: Xaa10 can be Asp; Xaa11 can be Asp; Xaa13 can be Pro; Xaa15 canbe Arg; Xaa16 can be Ala; Xaa17 can be Ala; Xaa18 can be His; Xaa19 canbe Pro; Xaa21 can be Trp; Xaa22 can be Phe; Xaa23 can be Phe; Xaa31 canbe Glu; Xaa32 can be Glu; Xaa34 can be Ile; Xaa35 can be Tyr; Xaa39 canbe Glu; Xaa40 can be Gly; Xaa43 can be Asn; and Xaa45 can be Phe, andmethods of using such compositions. In another embodiment, five or moreof the following of the following amino acid positions are defined asfollows: Xaa10 can be Asp; Xaa11 can be Asp; Xaa13 can be Pro; Xaa15 canbe Arg; Xaa16 can be Ala; Xaa17 can be Ala; Xaa18 can be His; Xaa19 canbe Pro; Xaa21 can be Trp; Xaa22 can be Phe; Xaa23 can be Phe; Xaa31 canbe Glu; Xaa32 can be Glu; Xaa34 can be Ile; Xaa35 can be Tyr; Xaa39 canbe Glu; Xaa40 can be Gly; Xaa43 can be Asn; and Xaa45 can be Phe. Inanother embodiment, 10 or more of the amino acids are defined asfollows: Xaa10 can be Asp; Xaa11 can be Asp; Xaa13 can be Pro; Xaa15 canbe Arg; Xaa16 can be Ala; Xaa17 can be Ala; Xaa18 can be His; Xaa19 canbe Pro; Xaa21 can be Trp; Xaa22 can be Phe; Xaa23 can be Phe; Xaa31 canbe Glu; Xaa32 can be Glu; Xaa34 can be Ile; Xaa35 can be Tyr; Xaa39 canbe Glu; Xaa40 can be Gly; Xaa43 can be Asn; and Xaa45 can be Phe. In yetanother embodiment, 15 or more of the amino acids are defined asfollows: Xaa10 can be Asp; Xaa11 can be Asp; Xaa13 can be Pro; Xaa15 canbe Arg; Xaa16 can be Ala; Xaa17 can be Ala; Xaa18 can be His; Xaa19 canbe Pro; Xaa21 can be Trp; Xaa22 can be Phe; Xaa23 can be Phe; Xaa31 canbe Glu; Xaa32 can be Glu; Xaa34 can be Ile; Xaa35 can be Tyr; Xaa39 canbe Glu; Xaa40 can be Gly; Xaa43 can be Asn; and Xaa45 can be Phe.

In a particular embodiment, the invention is directed to a compositioncomprising a polypeptide as defined by SEQ ID NO:1, such that, ifpresent, Xaa3 is Ser, Xaa2 is His, Xaa1 is Met, Xaa56 is Thr, Xaa57 isArg, and/or Xaa58 is Asp, and methods of using such compositions.

In another embodiment, the invention is directed to a compositioncomprising a polypeptide comprising the amino acid sequence:

Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala HisPro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr GlyGly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys MetCys Thr Arg Asp (SEQ ID NO:2), and methods of using such compositions.

In a particular embodiment, the present invention is directed to acomposition comprising a kallikrein binding polypeptide of 53-60 aminoacids comprising a Kunitz domain, wherein the Kunitz domain comprisesthe potential for disulfide bonds between cysteines at positions 5 and55; 14 and 38; and 30 and 51 (according to amino acid positionscorresponding to bovine pancreatic trypsin inhibitor (BPTI)), andfurther comprising:

-   -   amino acid number 13 selected from His and Pro;    -   amino acid number 16 selected from Ala and Gly;    -   amino acid number 17 selected from Ala, Asn, and Ser;    -   amino acid number 18 selected from His and Leu; and    -   amino acid number 19 selected from Gln, Leu, and Pro (SEQ ID        NO:23).

In a particular embodiment, the present invention is directed to acomposition comprising a kallikrein binding polypeptide of 53-60 aminoacids comprising a Kunitz domain, wherein the Kunitz domain comprisesthe potential for disulfide bonds between cysteines at positions 5 and55; 14 and 38; and 30 and 51 (according to amino acid positionscorresponding to bovine pancreatic trypsin inhibitor (BPTI)), andfurther comprising:

-   -   amino acid number 13 selected from His and Pro;    -   amino acid number 15 selected from Lys and Arg;    -   amino acid number 16 selected from Ala and Gly;    -   amino acid number 17 selected from Ala, Asn, and Ser;    -   amino acid number 18 selected from His and Leu; and    -   amino acid number 19 selected from Gln, Leu, and Pro,    -   amino acid number 31 is Glu;    -   amino acid number 32 selected from Glu and Gln;    -   amino acid number 34 selected from Ser, Thr, and Ile; and    -   amino acid number 39 selected from Gly, Glu, and Ala (SEQ ID        NO:24).

In a particular embodiment, the present invention is directed to acomposition comprising a kallikrein binding polypeptide of 53-60 aminoacids comprising a Kunitz domain, wherein the Kunitz domain comprises acysteine at each of positions 5 and 55; 14 and 38; and 30 and 51(according to amino acid positions corresponding to bovine pancreatictrypsin inhibitor (BPTI)), and further comprising:

-   -   amino acid number 13 selected from His and Pro;    -   amino acid number 15 selected from Lys and Arg;    -   amino acid number 16 selected from Ala and Gly;    -   amino acid number 17 selected from Ala, Asn, and Ser;    -   amino acid number 18 selected from His and Leu; and    -   amino acid number 19 selected from Gln, Leu, and Pro,    -   amino acid number 31 is Glu;    -   amino acid number 32 selected from Glu and Gln;    -   amino acid number 34 selected from Ser, Thr, and Ile; and    -   amino acid number 39 selected from Gly, Glu, and Ala (SEQ ID        NO:24).

In a particular embodiment, the Kunitz domain is selected from the groupconsisting of:

-   -   KKII/3 #1 (SEQ ID NO:24)    -   KKII/3 #2 (SEQ ID NO:25)    -   KKII/3 #3 (SEQ ID NO:26)    -   KKII/3 #4 (SEQ ID NO:27)    -   KKII/3 #5 (SEQ ID NO:28)    -   KKII/3 #6 (SEQ ID NO:29)    -   KKII/3 #7 (SEQ ID NO:30)    -   KKII/3 #8 (SEQ ID NO:31)    -   KKII/3 #9 (SEQ ID NO:32) and    -   KKII/3 #10 (SEQ ID NO:33)

as described in Table 1.

Each of the compositions described herein can be used in the methods ofthe invention. Further, the compounds described herein can be used inthe manufacture of a medicament or composition for the indications ormethods described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a simplified diagram of major multiple pathways and relatedevents involved in the contact activation system and systemicinflammatory response (SIR) that may arise in a patient subjected tosoft and bone tissue trauma such as that associated with a coronaryartery bypass grafting (CABG) procedure, especially when the CABGprocedure involves extra-corporeal blood circulation, such ascardiopulmonary bypass (CPB; Bypass Apparatus). Arrows indicateactivation from one component or event to another component or event inthe cascade. Arrows in both directions indicate activating effects ofcomponents or events in both directions. Broken arrows indicate likelyparticipation of one component or event in the activation of anothercomponent or event. Abbreviations are as follows: “tPA”=tissueplasminogen activator; “C5a”=a protein component of the complementsystem; “fXIIa”=activator protein of pre-kallikrein to form activekallikrein; “Extrinsic”=extrinsic coagulation system;“Intrinsic”=intrinsic coagulation system.

FIG. 2 shows a portion of a DNA and corresponding deduced amino acid fora kallikrein inhibitor (“KI”) polypeptide of the invention in plasmidpPIC-K503. The inserted DNA encodes the mata prepro signal peptide ofSaccharomyces cerevisiae (underlined) fused in frame to the aminoterminus of the PEP-1 KI polypeptide having the amino acid sequenceenclosed by the boxed area. The amino acid sequence of the PEP-1 KIpolypeptide shown in the boxed region is SEQ ID NO:2, and thecorresponding nucleotide coding sequence of the KI polypeptide is SEQ IDNO:3. The dashed arrows indicate the location and direction of two PCRprimer sequences in AOX regions that were used to produce sequencingtemplates. DNA sequence for the entire nucleotide sequence of the figurecomprises the structural coding sequence for the fusion protein and isdesignated SEQ ID NO:35. The double underlined portion of the sequenceindicates a diagnostic probe sequence. BstBI and EcoRI indicatelocations of their respective palindromic, hexameric, restrictionendonuclease sites in the sequence. Asterisks denote translational stopcodons.

FIG. 3 shows an alignment of amino acid sequences of the preferredembodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION

A description of preferred embodiments of the invention follows.

The invention is based on the discovery of kallikrein inhibitor (KI)polypeptides that inhibit plasma kallikrein with a specificity thatpermits their use in improved methods of preserving organs and tissues,such as pending a transplantation, and to corresponding methods. Theinvention also relates to reducing, inhibiting or preventing reperfusioninjury or damage in an organ or tissue that has been removed from itshost and compositions therefor.

Polypeptides Useful in the Invention

KI polypeptides useful in the invention comprise Kunitz domainpolypeptides. In one embodiment these Kunitz domains are variant formscomprising the looped structure of Kunitz domain 1 of humanlipoprotein-associated coagulation inhibitor (LACI) protein. LACIcontains three internal, well-defined, peptide loop structures that areparadigm Kunitz domains (Girard, T. et al., 1989. Nature, 338:518-520).The three Kunitz domains of LACI confer the ability to bind and inhibitkallikrein, although not with exceptional affinity. Variants of Kunitzdomain 1 of LACI described herein have been screened, isolated and bindkallikrein with enhanced affinity and specificity (see, for example,U.S. Pat. Nos. 5,795,865 and 6,057,287, incorporated herein byreference). An example of a preferred polypeptide useful in theinvention has the amino acid sequence defined by amino acids 3-60 of SEQID NO:2.

Kallikrein binding polypeptides can be used to target therapeutic ordiagnostic molecules to kallikrein in, for example, organ tissue, cells,or whole organisms. Such methods of targeted delivery for therapeutic ordiagnostic purposes would be known to one of skill in the art. Forexample, targeted kallikrein binding polypeptides could be used by oneof skill in the art to identify an organ that has been damaged by theeffects of kallikrien, or kallikrein can be targeted for the effects ofa particular therapeutic agent using kallikrein binding polypeptides ofthe invention.

Every polypeptide useful in the invention binds kallikrein. In preferredembodiments, the polypeptides are kallikrein inhibitors (KI) asdetermined using kallikrein binding and inhibition assays known in theart. The enhanced affinity and specificity for kallikrein of the variantKunitz domain polypeptides described herein provides the basis for theiruse in CPB and especially CABG surgical procedures to prevent or reduceperioperative blood loss and/or SIR in patients undergoing suchprocedures. The KI polypeptides used in the invention can have orcomprise the amino acid sequence of a variant Kunitz domain polypeptideoriginally isolated by screening phage display libraries for the abilityto bind kallikrein.

KI polypeptides useful in the methods and compositions of the inventioncomprise a Kunitz domain polypeptide comprising the amino acid sequence:

(SEQ ID NO: 1) Xaa1 Xaa2 Xaa3 Xaa4 Cys Xaa6 Xaa7 Xaa8 Xaa9 Xaa10 Xaa11Gly Xaa13 Cys Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Xaa20 Xaa21 Xaa22 Xaa23Xaa24 Xaa25 Xaa26 Xaa27 Xaa28 Xaa29 Cys Xaa31 Xaa32 Phe Xaa34 Xaa35 GlyGly Cys Xaa39 Xaa40 Xaa41 Xaa42 Xaa43 Xaa44 Xaa45 Xaa46 Xaa47 Xaa48Xaa49 Xaa50 Cys Xaa52 Xaa53 Xaa54 Cys Xaa56 Xaa57 Xaa58“Xaa” refers to a position in a peptide chain that can be any of anumber of different amino acids. For example, for the KI peptidesdescribed herein, Xaa10 can be Asp or Glu; Xaa11 can be Asp, Gly, Ser,Val, Asn, Ile, Ala or Thr; Xaa13 can be Pro, Arg, His, Asn, Ser, Thr,Ala, Gly, Lys or Gln; Xaa15 can be Arg, Lys, Ala, Ser, Gly, Met, Asn orGln; Xaa16 can be Ala, Gly, Ser, Asp or Asn; Xaa17 can be Ala, Asn, Ser,Ile, Gly, Val, Gln or Thr; Xaa18 can be His, Leu, Gln or Ala; Xaa19 canbe Pro, Gln, Leu, Asn or Ile; Xaa21 can be Trp, Phe, Tyr, His or Ile;Xaa31 can be Glu, Asp, Gln, Asn, Ser, Ala, Val, Leu, Ile or Thr; Xaa32can be Glu, Gln, Asp Asn, Pro, Thr, Leu, Ser, Ala, Gly or Val; Xaa34 canbe Ile, Thr, Ser, Val, Ala, Asn, Gly or Leu; Xaa35 can be Tyr, Trp orPhe; Xaa39 can be Glu, Gly, Ala, Ser or Asp. Amino acids Xaa6, Xaa7,Xaa8, Xaa9, Xaa20, Xaa24, Xaa25, Xaa26, Xaa27, Xaa28, Xaa29, Xaa41,Xaa42, Xaa44, Xaa46, Xaa47, Xaa48, Xaa49, Xaa50, Xaa52, Xaa53 and Xaa54can be any amino acid. Additionally, each of the first four and at lastthree amino acids of SEQ ID NO:1 can optionally be present or absent andcan be any amino acid, if present.

Peptides defined according to SEQ ID NO:1 form a set of polypeptidesthat bind to and inhibit kallikrein. The diversity of the KI's isincreased as the number of variable positions in the peptide sequence isincreased or as the number of amino acids possible at a variableposition increases. For example, in a preferred embodiment of theinvention, a KI polypeptide useful in the methods and compositions ofthe invention has the following variable positions: Xaa11 can be Asp,Gly, Ser or Val; Xaa13 can be Pro, Arg, His or Asn; Xaa15 can be Arg orLys; Xaa16 can be Ala or Gly; Xaa17 can be Ala, Asn, Ser or Ile; Xaa18can be His, Leu or Gln; Xaa19 can be Pro, Gln or Leu; Xaa21 can be Trpor Phe; Xaa31 is Glu; Xaa32 can be Glu or Gln; Xaa34 can be Ile, Thr orSer; Xaa35 is Tyr; and Xaa39 can be Glu, Gly or Ala.

A more specific embodiment of the claimed invention is defined by thefollowing amino acids at variable positions: Xaa10 is Asp; Xaa11 is Asp;Xaa13 can be Pro or Arg; Xaa15 is Arg; Xaa16 can be Ala or Gly; Xaa17 isAla; Xaa18 is His; Xaa19 is Pro; Xaa21 is Trp; Xaa31 is Glu; Xaa32 isGlu; Xaa34 can be Ile or Ser; Xaa35 is Tyr; and Xaa39 is Gly.

Also encompassed within the scope of the invention are peptides thatcomprise portions of the polypeptides described herein. For example,polypeptides could comprise binding domains for specific kallikreinepitopes. Such fragments of the polypeptides described herein would alsobe encompassed.

KI polypeptides useful in the methods and compositions described hereincomprise a Kunitz domain. A subset of the sequences encompassed by SEQID NO:1 are described by the following (where not indicated, “Xaa”refers to the same set of amino acids that are allowed for SEQ ID NO:1):

(SEQ ID NO: 36) Met His Ser Phe Cys Ala Phe Lys Ala Xaa10 Xaa11 GlyXaa13 Cys Xaa15 Xaa16 Xaa17 Xaa18 Xaa19 Arg Xaa21 Phe Phe Asn Ile PheThr Arg Gln Cys Xaa31 Xaa32 Phe Xaa34 Xaa35 Gly Gly Cys Xaa39 Gly AsnGln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp.Specific and particular examples of KI peptides useful in the inventiondescribed herein are as follows:

(amino acids 3-60 of SEQ ID NO: 2) Met His Ser Phe Cys Ala Phe Lys AlaAsp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe ThrArg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg PheGlu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 4) MetHis Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys Ala Asn His LeuArg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly GlyCys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met CysThr Arg Asp, (SEQ ID NO: 5) Met His Ser Phe Cys Ala Phe Lys Ala Asp AspGly His Cys Lys Ala Asn His Gln Arg Phe Phe Phe Asn Ile Phe Thr Arg GlnCys Glu Glu Phe Thr Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu SerLeu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 6) Met His SerPhe Cys Ala Phe Lys Ala Asp Asp Gly His Cys Lys Ala Asn His Gln Arg PhePhe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln Phe Thr Tyr Gly Gly Cys AlaGly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr ArgAsp, (SEQ ID NO: 7) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly HisCys Lys Ala Ser Leu Pro Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys GluGlu Phe Ile Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu GluGlu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 8) Met His Ser Phe CysAla Phe Lys Ala Asp Asp Gly His Cys Lys Ala Asn His Gln Arg Phe Phe PheAsn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly AsnGln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp,(SEQ ID NO: 9) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His CysLys Gly Ala His Leu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu GluPhe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu GluCys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 10) Met His Ser Phe Cys AlaPhe Lys Ala Asp Asp Gly Arg Cys Lys Gly Ala His Leu Arg Phe Phe Phe AsnIle Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn GlnAsn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ IDNO: 11) Met His Ser Phe Cys Ala Phe Lys Ala Asp Gly Gly Arg Cys Arg GlyAla His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe SerTyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys LysLys Met Cys Thr Arg Asp, (SEQ ID NO: 12) Met His Ser Phe Cys Ala Phe LysAla Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile PheThr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn ArgPhe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 13)Met His Ser Phe Cys Ala Phe Lys Ala Asp Val Gly Arg Cys Arg Gly Ala HisPro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr GlyGly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys MetCys Thr Arg Asp, (SEQ ID NO: 14) Met His Ser Phe Cys Ala Phe Lys Ala AspVal Gly Arg Cys Arg Gly Ala Gln Pro Arg Phe Phe Phe Asn Ile Phe Thr ArgGln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe GluSer Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 15) Met HisSer Phe Cys Ala Phe Lys Ala Asp Asp Gly Ser Cys Arg Ala Ala His Leu ArgTrp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly CysGly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys ThrArg Asp, (SEQ ID NO: 16) Met His Ser Phe Cys Ala Phe Lys Ala Glu Gly GlySer Cys Arg Ala Ala His Gln Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser LeuGlu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 17) Met His Ser PheCys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Gly Ala His Leu Arg Phe PhePhe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly GlyAsn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp,(SEQ ID NO: 18) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His CysArg Gly Ala Leu Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu GluPhe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu GluCys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 19) Met His Ser Phe Cys AlaPhe Lys Ala Asp Ser Gly Asn Cys Arg Gly Asn Leu Pro Arg Phe Phe Phe AsnIle Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn GlnAsn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ IDNO: 20) Met His Ser Phe Cys Ala Phe Lys Ala Asp Ser Gly Arg Cys Arg GlyAsn His Gln Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe SerTyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys LysLys Met Cys Thr Arg Asp, (SEQ ID NO: 21) Met His Ser Phe Cys Ala Phe LysAla Asp Gly Gly Arg Cys Arg Ala Ile Gln Pro Arg Trp Phe Phe Asn Ile PheThr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn ArgPhe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 22)Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Arg Cys Arg Gly Ala HisPro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr GlyGly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys MetCys Thr Arg Asp.FIG. 3 provides an amino acid sequence alignment of these sequences.

Other KI polypeptides useful in the present invention include:

(SEQ ID NO: 23) Arg Pro Asp Phe Cys Leu Glu Pro Pro Tyr Thr Gly Pro CysLys Ala Arg Ile Ile Arg Tyr Phe Tyr Asn Ala Lys Ala Gly Leu Cys Gln ThrPhe Val Tyr Gly Gly Cys Arg Ala Lys Arg Asn Asn Phe Lys Ser Ala Glu AspCys Met Arg Thr Cys Gly Gly Ala, (SEQ ID NO: 24) Met His Ser Phe Cys AlaPhe Lys Ala Asp Asp Gly His Cys Lys Ala Ser Leu Pro Arg Phe Phe Phe AsnIle Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn GlnAsn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ IDNO: 25) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys AlaAsn His Leu Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe SerTyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys LysLys Met Cys Thr Arg Asp, (SEQ ID NO: 26) Met His Ser Phe Cys Ala Phe LysAla Asp Asp Gly His Cys Lys Ala Asn His Gln Arg Phe Phe Phe Asn Ile PheThr Arg Gln Cys Glu Glu Phe Thr Tyr Gly Gly Cys Gly Gly Asn Gln Asn ArgPhe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 27)Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His Cys Lys Ala Asn HisGln Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Gln Phe Thr Tyr GlyGly Cys Ala Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys MetCys Thr Arg Asp, (SEQ ID NO: 28) Met His Ser Phe Cys Ala Phe Lys Ala AspAsp Gly His Cys Lys Ala Ser Leu Pro Arg Phe Phe Phe Asn Ile Phe Thr ArgGln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe GluSer Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 29) Met HisSer Phe Cys Ala Phe Lys Ala Asp Asp Gly His Cys Lys Ala Asn His Gln ArgPhe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly CysGly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys ThrArg Asp, (SEQ ID NO: 30) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp GlyHis Cys Lys Ala Asn His Gln Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln CysGlu Glu Phe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser LeuGlu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 31) Met His Ser PheCys Ala Phe Lys Ala Asp Asp Gly His Cys Lys Ala Asn His Gln Arg Phe PhePhe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ser Tyr Gly Gly Cys Gly GlyAsn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp,(SEQ ID NO: 32) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly His CysLys Ala Asn His Gln Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu GluPhe Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu GluCys Lys Lys Met Cys Thr Arg Asp, (SEQ ID NO: 33) Met His Ser Phe Cys AlaPhe Lys Ala Asp Asp Gly His Cys Lys Gly Ala His Leu Arg Phe Phe Phe AsnIle Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn GlnAsn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp, (SEQ IDNO: 34) Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys AlaIle Met Lys Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe IleTyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys LysLys Met Cys Thr Arg Asp.

These sequences are summarized in the following Table 1.

TABLE 1 Amino acid sequences of LACI(K1) variants selected for bindingto human plasma kallikrein. 13 16 17 18 19 31 32 34 39(a) KKII/3#1 H A SL P E E I E (SEQ ID NO: 24) KKII/3#2 P A N H L E E S G (SEQ ID NO: 25)KKII/3#3 H A N H Q E E T G (SEQ ID NO: 26) KKII/3#4 H A N H Q E Q T A(SEQ ID NO: 27) KKII/3#5 H A S L P E E I G (SEQ ID NO: 28) KKII/3#6 H AN H Q E E S G (SEQ ID NO: 29) KKII/3#7 H A N H Q E E S G (SEQ ID NO: 30)KKII/3#8 H A N H Q E E S G (SEQ ID NO: 31) KKII/3#9 H A N H Q E E S G(SEQ ID NO: 32) KKII/3#10 H G A H L E E I E (SEQ ID NO: 33) Consensus HA N H Q E E S/T G (a)Amino acid numbers of variegated residues. LACI(K1)(LACI residues 50-107 (SEQ ID NO: 32)) is 58 amino acids long with theP1 position being residue number 15 and fixed as lysine in thisinstance.

The polypeptides useful in the methods and compositions described hereinmay be made synthetically using any standard polypeptide synthesisprotocol and equipment. For example, the stepwise synthesis of a KIpolypeptide described herein may be carried out by the removal of anamino (N) terminal-protecting group from an initial (i.e.,carboxy-terminal) amino acid, and coupling thereto of the carboxyl endof the next amino acid in the sequence of the polypeptide. This aminoacid is also suitably protected. The carboxyl group of the incomingamino acid can be activated to react with the N-terminus of the boundamino acid by formation into a reactive group such as formation into acarbodiimide, a symmetric acid anhydride, or an “active ester” groupsuch as hydroxybenzotriazole or pentafluorophenyl esters. Preferredsolid-phase peptide synthesis methods include the BOC method, whichutilizes tert-butyloxycarbonyl as the a-amino protecting group, and theFMOC method, which utilizes 9-fluorenylmethloxycarbonyl to protect thea-amino of the amino acid residues. Both methods are well known to thoseof skill in the art (Stewart, J. and Young, J., Solid-Phase PeptideSynthesis (W.H. Freeman Co., San Francisco 1989); Merrifield, J., 1963.Am. Chem. Soc., 85:2149-2154; Bodanszky, M. and Bodanszky, A., ThePractice of Peptide Synthesis (Springer-Verlag, New York 1984), theentire teachings of these references is incorporated herein byreference). If desired, additional amino- and/or carboxy-terminal aminoacids may be designed into the amino acid sequence and added duringpolypeptide synthesis.

Alternatively, Kunitz domain polypeptides and KI polypeptides useful inthe compositions and methods of the invention may be produced byrecombinant methods using any of a number of cells and correspondingexpression vectors, including but not limited to bacterial expressionvectors, yeast expression vectors, baculovirus expression vectors,mammalian viral expression vectors, and the like. Kunitz domainpolypeptides and KI polypeptides useful in the compositions and methodsof the invention may also be produced transgenically using nucleic acidmolecules comprising a coding sequence for a Kunitz domain or KIpolypeptide described herein, wherein the nucleic acid molecule can beintegrated into and expressed from the genome of a host animal usingtransgenic methods available in the art. In some cases, it may benecessary or advantageous to fuse the coding sequence for a Kunitzdomain polypeptide or a KI polypeptide comprising the Kunitz domain toanother coding sequence in an expression vector to form a fusionpolypeptide that is readily expressed in a host cell. Preferably, thehost cell that expresses such a fusion polypeptide also processes thefusion polypeptide to yield a Kunitz domain or KI polypeptide useful inthe invention that contains only the desired amino acid sequence.Obviously, if any other amino acid(s) remain attached to the expressedKunitz domain or KI polypeptide, such additional amino acid(s) shouldnot diminish the kallikrein binding and/or kallikrein inhibitoryactivity of the Kunitz domain or KI polypeptide so as to preclude use ofthe polypeptide in the methods or compositions of the invention.

A preferred recombinant expression system for producing KI polypeptidesuseful in the methods and compositions described herein is a yeastexpression vector, which permits a nucleic acid sequence encoding theamino acid sequence for a KI polypeptide or Kunitz domain polypeptide tobe linked in the same reading frame with a nucleotide sequence encodingthe mata prepro leader peptide sequence of Saccharomyces cerevisiae,which in turn is under the control of an operable yeast promoter. Theresulting recombinant yeast expression plasmid may then be transformedby standard methods into the cells of an appropriate, compatible yeasthost, which cells are able to express the recombinant protein from therecombinant yeast expression vector. Preferably, a host yeast celltransformed with such a recombinant expression vector is also able toprocess the fusion protein to provide an active KI polypeptide useful inthe methods and compositions of the invention. A preferred yeast hostfor producing recombinant Kunitz domain polypeptides and KI polypeptidescomprising such Kunitz domains is Pichia pastoris.

As noted above, KI polypeptides that are useful in the methods andcompositions described herein may comprise a Kunitz domain polypeptidedescribed herein. Some KI polypeptides may have an additional flankingsequence, preferably of one to six amino acids in length, at the aminoand/or carboxy-terminal end, provided such additional amino acids do notsignificantly diminish kallikrein binding affinity or kallikreininhibition activity so as to preclude use in the methods andcompositions described herein. Such additional amino acids may bedeliberately added to express a KI polypeptide in a particularrecombinant host cell or may be added to provide an additional function,e.g., to provide a peptide to link the KI polypeptide to anothermolecule or to provide an affinity moiety that facilitates purificationof the polypeptide. Preferably, the additional amino acid(s) do notinclude cysteine, which could interfere with the disulfide bonds of theKunitz domain. Native examples of Kunitz domains exhibit disulfidebonds, e.g., BPTI contains disulfide bonds between cysteine residues atamino acid positions 5 and 55; 14 and 38; and 30 and 51

An example of a preferred Kunitz domain polypeptide useful in themethods and compositions of the invention has the amino acid sequence ofresidues 3-60 of SEQ ID NO:2. When expressed and processed in a yeastfusion protein expression system (e.g., based on the integratingexpression plasmid pHIL-D2), such a Kunitz domain polypeptide retains anadditional amino terminal Glu-Ala dipeptide from the fusion with themata prepro leader peptide sequence of S. cerevisiae. When secreted fromthe yeast host cell, most of the leader peptide is processed from thefusion protein to yield a functional KI polypeptide (also referred to as“PEP-1” or “DX88”) having the amino acid sequence of SEQ ID NO:2 (seeboxed region in FIG. 2).

Particularly preferred KI polypeptides useful in the methods andcompositions described herein have a binding affinity for kallikreinthat is on the order of 1000 times higher than that of aprotinin, whichis currently approved for use in CABG procedures to reduce blood loss.The surprisingly high binding affinities of such KI polypeptidesdescribed herein indicate that such KI polypeptides exhibit a highdegree of specificity for kallikrein to the exclusion of other moleculartargets (see Table 1, below). Thus, use of such polypeptides accordingto the invention reduces much of the speculation as to the possibletherapeutic targets. The lower degree of specificity exhibited by, forexample, aprotinin, leads to possible pleiotropic side effects andambiguity as to its therapeutic mechanism.

The polypeptides defined by, for example, SEQ ID NO:1 contain invariantpositions, e.g., positions 5, 14, 30, 51 and 55 can be Cys only. Otherpositions such as, for example, positions 6, 7, 8, 9, 20, 24, 25, 26,27, 28, 29, 41, 42, 44, 46, 47, 48, 49, 50, 52, 53 and 54 can be anyamino acid (including non-naturally occurring amino acids). In aparticularly preferred embodiment, one or more amino acids correspond tothat of a native sequence (e.g., LACI (SEQ ID NOS:32-34)). In apreferred embodiment, at least one variable position is different fromthat of the native sequence. In yet another preferred embodiment, theamino acids can each be individually or collectively substituted by aconservative or non-conservative amino acid substitution. Conservativeamino acid substitutions replace an amino acid with another amino acidof similar chemical structure and may have no affect on proteinfunction. Non-conservative amino acid substitutions replace an aminoacid with another amino acid of dissimilar chemical structure. Examplesof conserved amino acid substitutions include, for example, Asn→Asp,Arg→Lys and Ser→Thr. In a preferred embodiment, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and/or 21 of these aminoacids can be independently or collectively, in any combination, selectedto correspond to the corresponding position of SEQ ID NO:2.

Other positions, for example, positions 10, 11, 13, 15, 16, 17, 18, 19,21, 22, 23, 31, 32, 34, 35, 39, 40, 43 and 45, can be any of a selectedset of amino acids. Thus SEQ ID NO:1 defines a set of possiblesequences. Each member of this set contains, for example, a cysteine atpositions 5, 14, 30, 51 and 55, and any one of a specific set of aminoacids at positions 10, 11, 13, 15, 16, 17, 18, 19, 221, 22, 23, 31, 32,34, 35, 39, 40, 43 and 45. In a preferred embodiment, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 and/or 19 of these aminoacids can be independently or collectively, in any combination, selectedto correspond to the corresponding position of SEQ ID NO:2. The peptidepreferably has at least 80%, at least 85%, at least 90% or at least 95%identity to SEQ ID NO:2.

Methods and Compositions

The present invention is directed to methods for preserving organs andtissues comprising contacting the organ or tissue with a preservativesolution comprising a kallikrein inhibitor, such as those describedherein. The invention also relates to reducing, inhibiting or preventingreperfusion injury or damage in an organ or tissue that has been removedfrom its host comprising contacting the organ or tissue with akallikrein inhibitor. The preservative solutions of the invention can beused to preserve and/or protect organ tissue, or whole organs, when saidorgans or tissue are brought into contact with the solution. A specificembodiment of the invention is for the preservation of a human heart, orhuman myocardial tissue. Another embodiment of the invention is for thepreservation of a human lung or human lung tissue. Other organs, orparts thereof, that can be preserved according to the invention includekidney, liver, endothelial tissue, intestinal tissue, vascular tissue(e.g., an aorta graft), skin, and pancreas. The invention contemplatesthe use of the solutions to preserve mammalian tissue, organs or portionthereof. In addition, the solutions can be used to facilitatetransplantation of organs, e.g., by perfusion of the organ or tissueduring the transplantation procedure. The solution can also be used as acardioplegia solution in cardiac surgery. Preferably, the organ orportion thereof, is maintained in the appropriate solution at all times,particularly prior to the transplant procedure.

The solutions of the invention can be used to maintain viability of theorgan or tissue during storage, transplantation or other surgery. Theinvention includes a method of storing tissue or organs comprisingcontacting said tissue, organ or part thereof, with the solution of theinvention, such that the in vivo and/or in vitro viability is prolonged.The solutions permit maintenance of viability of heart or lung tissuefor up to 24 hours or more. Use of the solutions of the inventionresults in improved organ viability.

Alternatively or in addition, once removed from the donor, the organ orliving tissue may be placed in a preservation solution containing theinhibitor. In addition, the kallikrein inhibitor is also preferablyadministered to the transplant recipient just prior to, or concommitantwith, transplantation. In all cases, the inhibitor also can beadministered directly to the tissue at risk, as by injection to thetissue, or it may be provided systemically, either by oral or parenteraladministration, using any of the methods and formulations describedherein and/or known in the art.

In one embodiment, any commercially available preservation solution maybe used to advantage. Examples of such solutions include the Belzer UWsolution sold under the trademark VIASPAN, described in U.S. Pat. Nos.4,798,824, 4,873,230, 4,879,283, which are hereby incorporated byreference.

The preservation solution and perfusate composition described in theaforementioned patents includes, but is not limited to, the following:

TABLE 2 Amount in Substance 1 Liter K⁺-lactobionate 100 mmol KH₂PO₄ 25mmol MgSO₄ 5 mmol Raffinose 30 mmol Adenosine 5 mmol Glutathione 3 mmolInsulin 100 U Bactrim 0.5 mL Dexamethasone 8 mg Allopurinol 1 mMHydroxyethyl starch having a molecular 50 g weight of about 200,000 toabout 300,000 daltons and a degree of substitution of from about 0.4 to0.7

The solution is brought to pH 7.4 at room temperature with NaOH. Thefinal concentrations are Na=30.±0.5 mM, K⁺=120±5 mM, mOsm/liter=320±5.Bactrim=trimethoprim (16 mg/mL) and sulfamethoxazole (80 mg/mL). Thehydroxyethyl starch can be present in the range of from about 3 to about8%.

This solution typically provides for a 72 hour preservation of thepancreas, 48 hour preservation for the kidney and at least 24 hourpreservation for the liver. U.S. Pat. No. 5,145,771, incorporated hereinby reference, described the organ preservation solution known as the“Carolina Solution,” which is also useful in the present invention. Therinse or preservation solution composition described in theaforementioned patent includes, but is not limited to, the components inabout the concentration ranges set forth in Table 3 below.

TABLE 3 Concentration Ranges in 1 Liter 10% modified hydroxyethyl starch30 g/L to 100 g/L NaCl 85 mM to 145 mM KCl 3 mM to 6 mM CaCl₂ 1.0 mM to1.6 mM KH₂PO₄ 0.7 mM to 1.3 mM MgSO₄ 0.9 mM to 1.5 mM Allopurinol 0.05mM to 5.0 mM Desferrioxamine 0.02 mM to 2.0 mM Glutathione 0.5 mM to10.0 mM Nicardipene 0.1 .mu.M to 5.0 .mu.M Adenosine 0.1 mM to 5.0 mMFructose 1.0 mM to 50.0 mM Glucose 1.0 mM to 50.0 mM Insulin 5 U/L to250 U/L Mops 2 mM 40 mM

One specific embodiment is prepared with the components in the amountsset forth in Table 4 below in accordance with the instructions set forthbelow.

TABLE 4 Components of 1 Liter Rinse Solution 500 mL Distilled DeionizedWater 50 g/L 10% modified hydroxyethyl starch 115 mM NaC1 6.7 g 5 mM KCl0.37 g 1.30 mM CaCl₂ 0.19 g 1 mM KH₂PO₄ 0.14 g 1.2 mM MgSO₄ 0.15 g 1 mMAllopurinol 0.14 g 1 mM Desferrioxamine 0.65 g 3 mM Glutathione 0.92 g 2.mu.M Nicardipene 0.80 mg 1 mM Adenosine 0.32 g 10 mM Fructose 1.8 g 10mM Glucose 1.8 g 100 U/L Insulin 100 units 20 mM Mops 4.2 g

In one embodiment this solution can be prepared as follows: using a 500mL volumetric flask, measure 500 mL of 10% (weight/volume) hydroxyethylstarch solution and pour into a 1 L beaker. Add 400 mL of doubledistilled water and stir vigorously using a magnetic stir bar. Add therest of the components one at a time. After all components are added,adjust the pH to 6.5 with 1-2 mL 5N NaOH. The solution should be stirredfor at least thirty minutes. Transfer the solution to a 1 L volumetricflask and bring to 1 L final volume. Filter to remove any undissolvedparticles.

Still another embodiment is exemplified by Table 5 below.

TABLE 5 Concentration Ranges in 1 Liter NaCl  85 mM to 145 mM  KCl   3mM to   6 mM CaCl₂ 1.0 mM to 1.6 mM KH₂PO₄ 0.7 mM to 1.3 mM MgSO₄ 0.9 mMto 1.5 mM Adenosine 0.12 mM  to 1.2 mM

A composition according to Table 5 above may optionally include one,several, or all of the further ingredients specified in Table 3 above.Preferably, the composition includes at least one antioxidant. Thus, onespecific embodiment of a composition is set forth in Table 6 below:

TABLE 6 Components of 1 Liter Rinse Solution 500 mL Distilled DeionizedWater 115 mM NaCl  6.7 g 5 mM KCl 0.37 g 1.30 mM CaCl₂ 0.19 g 1 mMKH₂PO₄ 0.14 g 1.2 mM MgSO₄ 0.15 g 1 mM Allopurinol 0.14 g 1 mMDesferrioxamine 0.65 g 3 mM Glutathione 0.92 g .12 mM Adenosine 0.038 g 

Preferred compositions may further comprise one or more pharmaceuticallyacceptable buffers, carriers, antioxidants, protease inhibitors, orother anti-ischemia agents.

Compositions useful in the methods of the invention comprise any of theKunitz domain polypeptides or KI polypeptides comprising such Kunitzdomain polypeptides described herein. Particularly preferred are KIpolypeptides comprising a Kunitz domain polypeptide having a 58-aminoacid sequence of amino acids 3-60 of SEQ ID NO:2. An example of such aparticularly preferred KI polypeptide useful in the methods andcompositions of the invention is the PEP-1 KI polypeptide having the60-amino acid sequence of SEQ ID NO:2. A nucleotide sequence encodingthe amino acid sequence of SEQ ID NO:2 is provided in SEQ ID NO:3 (see,e.g., nucleotides 309-488 in FIG. 2). It is understood that based on theknown genetic code, the invention also provides degenerate forms of thenucleotide sequence of SEQ ID NO:3 by simply substituting one or more ofthe known degenerate codons for each amino acid encoded by thenucleotide sequence. Nucleotides 7-180 of SEQ ID NO:3, and degenerateforms thereof, encode the non-naturally occurring Kunitz domainpolypeptide having the 58-amino acid sequence of amino acids 3-60 of SEQID NO:2.

Concentration Considerations for KI Polypeptides

Several considerations regarding dosing with a KI polypeptide in methodsof the invention may be illustrated by way of example with therepresentative PEP-1 KI polypeptide of the invention having the aminosequence of SEQ ID NO:2 (molecular weight of 7,054 Daltons).

Table 7, below, provides a comparison of the affinity (K_(i,app)) of thePEP-1 KI polypeptide for kallikrein and eleven other known plasmaproteases.

TABLE 7 Protease Substrate PEP-1 K_(i,app) (pM) Aprotinin K_(i,app) (pM)human plasma kallikrein 44 3.0 × 10⁴ human urine kallikrein   >1 × 10⁸4.0 × 10³ porcine pancreatic kallikrein  2.7 × 10⁷ 550 human C1r,activated >2.0 × 10⁸ 1.0 × 10⁷ human C1s, activated >2.0 × 10⁷ >1.0 ×10⁸  human plasma factor XIa   1.0 × 10⁴ ND human plasma factorXIIa >2.0 × 10⁷ >1.0 × 10⁸  human plasmin  1.4 × 10⁵ 894 humanpancreatic trypsin   >2 × 10⁷ ND human pancreatic >2.0 × 10⁷ 7.3 × 10⁵chymotrypsin human neutrophil elastase >2.0 × 10⁷ 1.7 × 10⁶ human plasmathrombin >2.0 × 10⁷ >1.0 × 10⁸  ND = not determined

Clearly, the PEP-1 KI polypeptide is highly specific for human plasmakallikrein. Furthermore, the affinity (K_(i,app)) of PEP-1 forkallikrein is 1000 times higher than the affinity of aprotinin forkallikrein: the K_(i,app) of PEP-1 for kallikrein is about 44 pM (Table1), whereas the K_(i,app) of aprotinin for kallikrein is 30,000 pM.Thus, a dose of PEP-1 could be approximately 1000 times lower than thatused for aprotinin on a per mole basis. However, consideration ofseveral other factors may provide a more accurate estimation of the doseof PEP-1 required in practice. Such factors include the amount ofkallikrein activated upon organ removal from a particular patient, andwill be recognized by the skilled artisan.

The invention will be further described with reference to the followingnon-limiting examples. The teachings of all the patents, patentapplications and all other publications and websites cited herein areincorporated by reference in their entirety.

EXEMPLIFICATION Example 1 A Representative KI Polypeptide

A KI polypeptide (PEP-1) useful in the compositions and methods of theinvention was identified as a kallikrein binding polypeptide displayedon a recombinant phage from a phage display library. PEP-1 has thefollowing amino acid sequence: Glu Ala Met His Ser Phe Cys Ala Phe LysAla Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile PheThr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn ArgPhe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp (SEQ ID NO:2).The molecular weight of PEP-1 is 7,054 Daltons.

The nucleotide sequence (SEQ ID NO:3) of the recombinant phage DNAencoding the PEP-1 amino acid sequence (amino acids 3-60 of SEQ ID NO:2)was isolated and sequenced by standard methods determined from therecombinant phage DNA. PEP-1 was produced in amounts useful for furthercharacterization as a recombinant protein in His4⁻ phenotype host cellsof yeast strain Pichia pastoris.

Example 2 Construction of a Recombinant Plasmid to Express KIPolypeptides

The initial plasmid, pHIL-D2, is ampicillin resistant and contains awild-type allele of His4 from P. pastoris. The final DNA sequencecomprising the coding sequence for the mata Prepro-PEP-1 fusion proteinin the recombinant expression plasmid pPIC-K503 is shown in FIG. 2. TheDNA sequence of pHIL-D2 was modified to produce pPIC-K503, as follows:

-   -   1. The BstBI site in the 3′ AOX1 region of pHIL-D2, located        downstream of the His4 gene, was removed by partial restriction        digestion, fill-in, and ligation, altering the sequence from        TTCGAA (SEQ ID NO:23) to TTCGCGAA (SEQ ID NO:24). This        modification was made to facilitate and direct the cloning of        the expression cassette into the plasmid.    -   2. The AatII site bearing the bla gene located downstream of        His4 was removed by restriction digestion, fill-in, and ligation        modifying the sequence from GACGTC (SEQ ID NO:25) to GACGTACGTC        (SEQ ID NO:26). This modification was made to facilitate the        cloning of expression cassettes having AatII sites into the        plasmid. The DNA encoding PEP-1 was synthesized based on the        nucleotide sequence from the original kallikrein-binding display        phage and consisted of 450 base pairs (bp). The final DNA        sequence of the insert in the pHIL-D2 plasmid is flanked by a 5′        AOX1 sequence and a 3′ AOX1 sequence (portions of which are        shown in FIG. 2) and encode a fusion protein comprising the mata        prepro signal peptide of S. cerevisiae fused to the structural        coding sequence for the PEP-1 KI polypeptide. The signal peptide        was added to facilitate the secretion of PEP-1 from the yeast        host cells. The oligonucleotides to form the insert were        synthesized and obtained commercially (Genesis Labs, The        Woodlands, Tex.), and the insert was generated by polymerase        chain reaction (PCR). The linked synthetic DNA encoding the mata        prepro/PEP-1 fusion protein was then incorporated by ligation        into the modified pHIL-D2 plasmid between the BstBI and EcoRI        sites.

The ligation products were used to transform Escherichia coli strain XL1Blue. A PCR assay was used to screen E. coli transformants for thedesired plasmid construct. DNA from cell extracts was amplified by PCRusing primers containing the 5′ AOX1 and 3′ AOX1 sequences (see aboveand FIG. 2). PCR products of the correct number of base pairs weresequenced. In addition, approximately 20-50 by on either side of thecloning sites were sequenced, and the predicted sequence was obtained.The final DNA sequence of the insert in the pHIL-D2 plasmid (to yieldplasmid pPIC-K503) is shown in FIG. 2 along with portions of flanking 5′and 3′ AOX1 sequences and corresponding amino acid sequence of thefusion protein comprising the mata prepro signal peptide of S.cerevisiae fused to the structural coding sequence for the PEP-1 KIpolypeptide. A transformant with the desired expression plasmidconstruct, plasmid pPIC-K503, was selected for preparing yeast celllines for routine production of PEP-1.

Example 3 Manufacture of PEP-1 from Recombinant Yeast Cell Line

Spheroplasts of P. pastoris GS115 having the His4⁻ phenotype weretransformed with the expression plasmid pPIC-K503 (above) followinglinearization of the plasmid at the SacI site and homologousrecombination of the plasmid DNA into the host 5′ AOX1 locus. Thephenotype of the production strain is His4⁺. The entire plasmid wasinserted into the 5′ AOX1 genomic sequence of the yeast.

Isolates from the transformation were screened for growth in the absenceof exogenous histidine with methanol as the sole carbon source. Greaterthan 95% of the transformants retained the wild-type ability to growwith methanol as the sole carbon source, thereby demonstrating that theplasmid had been inserted into the host genome by homologousrecombination rather than transplacement. These transformants did notrequire exogenous histidine for growth, thereby demonstrating that theplasmid had integrated into the host genome. Selected colonies werecloned. Small culture expression studies were performed to identifyclones secreting the highest levels of active PEP-1 into the culturemedium. PEP-1 secretion levels in clarified culture supernatantsolutions were quantified for PEP-1 levels by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and evaluated forkallikrein inhibition. A yeast clone was selected for PEP-1 productionbased on its high level of PEP-1 expression among cultures sampled.

Master and working cell banks of P. pastoris producing PEP-1 wereprepared commercially (MDS Pharma Services, Bothell, Wash.). A standardproduction of PEP-1 in yeast comprised three steps as follows: (1)preparation of the seed culture, (2) fermentation, and (3) recovery ofthe culture.

The seed culture step consisted of the inoculation of six flasks (300 mLeach) containing sterile inoculum broth (yeast nitrogen base, potassiumphosphate, and glycerol, pH=5) with the contents of a single vial of aworking cell bank of P. pastoris producing PEP-1. Flasks were inoculatedin an orbital shaker (300 rpm) for approximately 13 hours at 30° C.±2°C.

Fermentations were performed in a closed 100 liter Braun fermenterfilled with sterile broth. Each fermentation was initiated with thetransfer of the contents of the six seed culture flasks to thefermenter. After approximately 24 hours, the glycerol in the fermenterbecame exhausted and additional glycerol was added for approximately 8additional hours.

A mixed feed phase, which lasted approximately 83 hours, was theninitiated by the addition of a glycerol and methanol feed. At the end ofthis time, the fermentation was terminated, and the fermenter contentswere diluted with purified water. The purification and processing ofPEP-1 consisted of five steps as follows: (1) expanded bedchromatography, (2) cation exchange chromatography, (3) hydrophobicinteraction chromatography (HIC), (4) ultrafiltration and diafiltration,and (5) final filtration and packaging.

The initial purification step consisted of expanded bed chromatography.The diluted fermenter culture was applied to the equilibrated columnpacked with Streamline SP resin (Amersham Pharmacia Streamline 200chromatography column, Amersham Pharmacia, Piscataway, N.J.). The columnwas then washed (50 mM acetic acid, pH=3.0-3.5) in an up-flow mode toflush the yeast cells from the expanded bed. The top adaptor was raisedabove the expanded bed enhance washing. The flow was stopped and the bedwas allowed to settle. The adaptor was moved down so that it wasslightly above the settled bed. The direction of the flow was reversed.The effluent was collected. Washing was continued in a downward modeusing 50 mM sodium acetate, pH 4.0. The effluent was collected. PEP-1was eluted from the column using 50 mM sodium acetate, pH 6.0. Theeluate was collected in a 50 liter container. The eluate was thenfiltered through a 0.22 m filter into a clean container located in thepurification site. Additional samples were collected for thedetermination of PEP-1 concentration. A cation exchange chromatographystep was then performed using the filtered eluate from the expanded bedcolumn. PEP-1 was eluted from the column using 15 mM trisodium citrate,pH 6.2.

Additional proteins were removed from the PEP-1 preparation byhydrophobic interaction chromatography (HIC). Prior to HIC, the eluatefrom the cation exchange column was diluted with ammonium sulfate. Theeluate was applied to the column, and the PEP-1 was eluted usingammonium sulfate (0.572 M) in potassium phosphate (100 mM), pH 7.0. Theeluate was collected in fractions based on A280 values. All fractionswere collected into sterile, pre-weighed PETG bottles.

Selected fractions were pooled into a clean container. The pool wasconcentrated by ultrafiltration. The concentrated PEP-1 preparation wasimmediately diafiltered against ten volumes of PBS, pH 7.0.

A final filtration step was performed prior to packaging in order tominimize the bioburden in the bulk PEP-1. The bulk solution was filteredthrough a 0.22 m filter and collected into a sterile, pre-weighed PETGbottle. A sample was removed for lot release testing. The remainder ofthe bulk was dispensed aseptically into sterile PETG bottles and storedat −20° C.

Example 4 Kallikrein Inhibition Assay

A kinetic test was used to measure inhibitory activity of KIpolypeptides, such as PEP-1. The kinetic assay measures fluorescencefollowing kallikrein-mediated cleavage of a substrate,prolylphenylalanylarginyl amino methyl coumarin. A known amount ofkallikrein was incubated with a serially diluted KI polypeptidereference standard or serially diluted KI polypeptide test samples, in asuitable reaction buffer on a microtiter plate. Each sample was run intriplicate. The substrate solution was added, and the plate readimmediately using an excitation wavelength of 360 nm and an emissionwavelength of 460 nm. At least two each of the reference standard andsample curves were required to have an R-squared value of 0.95 to beconsidered valid.

Example 5 Organ Preservation

HUVEC at confluence were washed in PBS and further incubated at 4degrees for 24-48 hours in a serum free medium (SFM). After coldstorage, cells were washed several times with PBS, and kallikrein (0.125U) and the specific kallikrein substrate S2302 were added to the cells.Changes in optical density were recorded. For light microscopyevaluation of cell-bound PEP-1, after cold storage, HUVEC were treatedwith PEP-1, formalin fixed and treated with rabbit anti-PEP-1 andperoxidase conjugated anti-rabbit IgG. The ability of HUVEC to producekallikrein was also evaluated on cell surface and in the supernatants ofcells maintained at 37° C. Kallikrein activity was 380±19 A.U. insupernatants of HUVEC maintained at 37° C.; no activity was measurableon the surface of the same cells. At light microscopy evaluation therewas significant binding of PEP-1 to the surface of HUVEC cold treatedfor 24 hours. The maximum of the binding was obtained by incubatingcells in presence of PEP-1 (5 mg/ml). Cell-bound PEP-1 retained theability to inhibit exogenous kallikrein. These results indicate thatPEP-1 binds to endothelial cells, maintaining its kallikrein inhibitoryactivity. Therefore it can be used to detect and modulate kinin-mediateddamage on the vascular surface.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A method for reducing kallikrein activity in aremoved organ or tissue, the method comprising: contacting the organ ortissue, ex vivo, with an effective amount of a polypeptide comprisingamino acids 3-60 of SEQ ID NO:
 2. 2. The method of claim 1, wherein thepolypeptide comprises SEQ ID NO:
 2. 3. The method of claim 1, whereinthe organ or tissue is selected from the group consisting of heart,lung, kidney, pancreas, liver, intestine, endothelial tissue, vasculartissue and skin.
 4. The method of claim 1, wherein the polypeptideconsists of amino acids 3-60 of SEQ ID NO:
 2. 5. The method of claim 1,wherein the polypeptide consists of SEQ ID NO:
 2. 6. A method forreducing reperfusion injury in a removed organ or tissue, the methodcomprising: contacting the organ or tissue, ex vivo, with an effectiveamount of a polypeptide comprising amino acids 3-60 of SEQ ID NO:
 2. 7.The method of claim 6, wherein the polypeptide comprises SEQ ID NO: 2.8. The method of claim 6, wherein the organ or tissue is selected fromthe group consisting of heart, lung, kidney, pancreas, liver, intestine,endothelial tissue, vascular tissue and skin.
 9. The method of claim 6,wherein the polypeptide consists of amino acids 3-60 of SEQ ID NO: 2.10. The method of claim 6, wherein the polypeptide consists of SEQ IDNO: 2.